SEVERE BURN-INDUCED MITOCHONDRIAL RECRUITMENT OF CALPAIN CAUSES ABERRANT MITOCHONDRIAL DYNAMICS AND HEART DYSFUNCTION.

ABSTRACT: Mitochondrial damage is an important cause of heart dysfunction after severe burn injury. However, the pathophysiological process remains unclear. This study aims to examine the mitochondrial dynamics in the heart and the role of µ-calpain, a cysteine protease, in this scenario. Rats were subjected to severe burn injury treatment, and the calpain inhibitor MDL28170 was administered intravenously 1 h before or after burn injury. Rats in the burn group displayed weakened heart performance and decreased mean arterial pressure, which was accompanied by a diminishment of mitochondrial function. The animals also exhibited higher levels of calpain in mitochondria, as reflected by immunofluorescence staining and activity tests. In contrast, treatment with MDL28170 before any severe burn diminished these responses to a severe burn. Burn injury decreased the abundance of mitochondria and resulted in a lower percentage of small mitochondria and a higher percentage of large mitochondria. Furthermore, burn injury caused an increase in the fission protein DRP1 in the mitochondria and a decrease in the inner membrane fusion protein OPA1. Similarly, these alterations were also blocked by MDL28170. Of note, inhibition of calpain yielded the emergence of more elongated mitochondria along with membrane invagination in the middle of the longitude, which is an indicator of the fission process. Finally, MDL28170, administered 1 h after burn injury, preserved mitochondrial function and heart performance, and increased the survival rate. Overall, these results provided the first evidence that mitochondrial recruitment of calpain confers heart dysfunction after severe burn injury, which involves aberrant mitochondrial dynamics.

Molecular Mechanism of Mouse Uterine Smooth Muscle Regulation on Embryo Implantation.

Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.

CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice.

Macrophages are essential for wound repair after myocardial infarction (MI). CD226, a member of immunoglobulin superfamily, is expressed on inflammatory monocytes, however, the role of CD226 in infarct healing and the effect of CD226 on macrophage remain unknown. Methods: Wild type and CD226 knockout (CD226 KO) mice were subjected to permanent coronary ligation. CD226 expression, cardiac function and ventricular remodeling were evaluated. Profile of macrophages, myofibroblasts, angiogenesis and monocytes mobilization were determined. Results: CD226 expression increased in the infarcted heart, with a peak on day 7 after MI. CD226 KO attenuated infarct expansion and improved infarct healing after MI. CD226 deletion resulted in increased F4/80+ CD206+ M2 macrophages and diminished Mac-3+ iNOS+ M1 macrophages accumulation in the infarcted heart, as well as enrichment of α-smooth muscle actin positive myofibroblasts and Ki67+ CD31+ endothelial cells, leading to increased reparative collagen deposition and angiogenesis. Furthermore, CD226 deletion restrained inflammatory monocytes mobilization, as revealed by enhanced retention of Ly6Chi monocytes in the spleen associated with a decrease of Ly6Chi monocytes in the peripheral blood, whereas local proliferation of macrophage in the ischemic heart was not affected by CD226 deficiency. In vitro studies using bone marrow-derived macrophages showed that CD226 deletion potentiated M2 polarization and suppressed M1 polarization. Conclusion: CD226 expression is dramatically increased in the infarcted heart, and CD226 deletion improves post-infarction healing and cardiac function by favoring macrophage polarization towards reparative phenotype. Thus, inhibition of CD226 may represent a novel therapeutic approach to improve wound healing and cardiac function after MI.

A New Chimeric Natriuretic Peptide, CNAAC, for the Treatment of Left Ventricular Dysfunction after Myocardial Infarction.

An innovative natriuretic peptide analog named CNAAC (structurally consisting of the C-terminus and ring of ANP and the N-terminus of CNP) that has been shown to exhibit potent vasodilatory, diuretic, and hypotensive effects in our previous study was evaluated for the treatment of left ventricular dysfunction following myocardial infarction. The temporal relaxation effect and metabolic status of CNAAC were determined. A myocardial ischemic model was established. Rats were randomly divided into Sham, MI, MI-ANP, MI-CNP, MI-VNP, and MI-CNAAC groups. Humoral factors were measured; echocardiography and hemodynamics methods were employed to assess the cardiac function at the fourth week after modeling. The results showed that CNAAC had a potent relaxant effect and longer duration of action than ANP, CNP, or VNP. The stability of CNAAC in blood was higher than other three NPs. Four weeks of NP administration ameliorated diastolic and systolic dysfunction, the hypertrophic index, myocardial fibrosis, and infarct size; it also restored the abnormal changes in humoral factors. These results demonstrate that CNAAC has a potent cardioprotective effect against left ventricular dysfunction after myocardial infarction. The results may lay the foundation for the clinical application of this newly designed NP chimera in the treatment and prevention of heart failure.

Performance of urinary neutrophil gelatinase-associated lipocalin, clusterin, and cystatin C in predicting diabetic kidney disease and diabetic microalbuminuria: a consecutive cohort study.

BACKGROUND: Tubular biomarkers have been regarded as emerging and promising markers for early diagnosis of diabetic kidney disease (DKD). The study was to determine the diagnostic capabilities of tubular biomarkers (urinary neutrophil gelatinase-associated lipocalin [NGAL], clusterin, and cystatin C) for DKD and diabetic microalbuminuria, and whether or not the tubular biomarkers appear earlier than microalbuminuria. METHODS: In this consecutive cohort study, 146 type 2 diabetes mellitus (T2DM) patients with a disease duration of ≥6 years were enrolled. Thirty age- and gender-matched subjects without any systemic diseases were recruited as the control group. Urinary samples collected before treatment were tested for NGAL, clusterin, and cystatin C. RESULTS: The levels of biomarkers were higher in patients with DKD (p < 0.001); and positively correlated with the urinary albumin creatinine ratio (UACR; p < 0.001). With respect to the diagnosis of DKD, the areas under the receiver operating characteristic curve (AUCs) for urinary NGAL, clusterin, and cystatin C were 0.816 (95% confidence interval [CI], 0.741-0.891), 0.775 (95% CI: 0.694-0.857), and 0.803 (95% CI: 0.722-0.884), respectively. The levels of urinary NGAL and cystatin C in the normoalbuminuria group (UACR <30 mg /g•Cr) were elevated compared with the control group, unlike urinary clusterin. There was no statistical difference in the levels of the three biomarkers between groups with different levels of haemoglobin A1C (HbA1c). The diagnostic AUCs for urinary NGAL, clusterin, and cystatin C in patients with diabetic microalbuminuria were 0.841 (95% CI: 0.775-0.907), 0.783(95% CI: 0.710-0.856), and 0.805 (95% CI: 0.733-0.877), respectively. CONCLUSIONS: Urinary NGAL, clusterin, and cystatin C may be promising biomarkers for diagnosing DKD and diabetic microalbuminuria. It is possible that urinary NGAL and cystatin C increase before the onset of microalbuminuria in T2DM patients.

κ-Opioid Receptor Stimulation Improves Endothelial Function via Akt-stimulated NO Production in Hyperlipidemic Rats.

This study was designed to investigate the effect of U50,488H (a selective κ-opioid receptor agonist) on endothelial function impaired by hyperlipidemia and to determine the role of Akt-stimulated NO production in it. Hyperlipidemic model was established by feeding rats with a high-fat diet for 14 weeks. U50,488H and nor-BNI (a selective κ-opioid receptor antagonist) were administered intraperitoneally. In vitro, the involvement of the PI3K/Akt/eNOS pathway in the effect of U50,488H was studied using cultured endothelial cells subjected to artificial hyperlipidemia. Serum total cholesterol and low-density lipoprotein cholesterol concentrations dramatically increased after high-fat diet feeding. Administration of U50,488H significantly alleviated endothelial ultrastructural destruction and endothelium-dependent vasorelaxation impairment caused by hyperlipidemia. U50,488H also increased Akt/eNOS phosphorylation and serum/medium NO level both in vivo and in vitro. U50,488H increased eNOS activity and suppressed iNOS activity in vivo. The effects of U50,488H were abolished in vitro by siRNAs targeting κ-opioid receptor and Akt or PI3K/Akt/eNOS inhibitors. All effects of U50,488H were blocked by nor-BNI. These results demonstrate that κ-opioid receptor stimulation normalizes endothelial ultrastructure and function under hyperlipidemic condition. Its mechanism is related to the preservation of eNOS phosphorylation through activation of the PI3K/Akt signaling pathway and downregulation of iNOS expression/activity.

Lacidipine attenuates TNF-α-induced cardiomyocyte apoptosis.

This study was designed to investigate whether lacidipine elicited a protective role on cardiomyocyte against apoptosis induced by TNF-α. Neonatal rat cardiomyocytes were randomly assigned into different groups. TUNEL staining was utilized to detect apoptosis, and caspase-3 and caspse-12 were determined. To explore the underlying mechanism, Z-ATAD-FMK (a selective caspase-12 inhibitor) was used to identify the key molecule involved. TNF-α increased caspase-3 expression, which was mediated by increased caspase-12 expression. In the meantime, apoptosis was significantly induced by TNF-α. Lacidipine lowered caspase-12 and caspase-3 expression, and cardiomyocyte apoptosis induced by TNF-α. The results suggest that lacidipine attenuates TNF-α -induced apoptosis via inhibition of caspase-12 and caspase-3 successively.

Anti-arrhythmic effect of verapamil is accompanied by preservation of cx43 protein in rat heart.

The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.

κ-opioid receptor activation prevents against arrhythmias by preserving Cx43 protein via alleviation of intracellular calcium.

κ-opioid receptor (κ-OR) activation with U50,488H, a selective κ-OR agonist, has been previously demonstrated to prevent against cardiac arrhythmias via stabilizing the synthesis and degradation of an integral membrane protein, Cx43, in gap junctions. However, the exact prevention mechanism remains unclear. The present study tested the hypothesis that the kappa OR agonist U50,488H mediates the prevention of arrhythmia through the regulation of intracellular calcium leading to the preservation of Cx43 protein. By performing electrocardiogram monitoring and immunoblotting in isolated Langendorff-perfused rat hearts, high concentrations of calcium-perfused rat hearts exhibited increased cardiac arrhythmias. Diminished expression of Cx43 protein was observed. The utilization of a whole-cell patch clamp technique revealed that U50,488H inhibited L-type calcium current in single ventricular myocytes in a dose-dependent manner. These effects were blocked by nor-binaltorphimine, potent and selective κ-OR antagonists. Administration of U50,488H before myocardial ischemia resulted in an attenuated of total arrhythmia scores. The attenuation effect was blocked by nor-binaltorphimine. The attenuation effect was antagonized both by Bay K8644, a L-type calcium channel agonist, and also by the Cx43 uncoupler heptanol. Finally, immunoblotting data demonstrated that the preservation of Cx43 protein conferred by U50,488H was reversed in the presence of Bay K8644. In summary, the present study demonstrates κ-OR activation with U50,488H may confer antiarrhythmic effects via modulation of the calcium-Cx43 pathway.

Vasculoprotective effect of U50,488H in rats exposed to chronic hypoxia: role of Akt-stimulated NO production.

Impairment of pulmonary endothelium function in the pulmonary artery is a direct result of chronic hypoxia. This study is to investigate the vasculoprotective effects of U50,488H (a selective κ-opioid receptor agonist) and its underlying mechanism in hypoxia-induced pulmonary artery endothelial functional injury. Chronic hypoxia was simulated by exposing the rats to 10% oxygen for 2 wk. After hypoxia, right ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI) were measured. The pulmonary vascular dysfunction, effect of nitric oxide synthase inhibitor (l-NAME) on the relaxation of U50,488H, and level of nitric oxide (NO) were determined. In vitro, the signaling pathway involved in the anti-apoptotic effect of U50,488H was investigated. Cultured endothelial cells were subjected to simulated hypoxia, and cell apoptosis was determined by TUNEL staining. U50,488H (1.25 mg/kg) significantly reduced RVP and RVHI in hypoxia. U50,488H markedly improved both pulmonary endothelial function (maximal vasorelaxation in response to ACh: 74.9 ± 1.8%, n = 6, P <0.01 vs. hypoxia for 2 wk group) and increased total NO production (1.65 fold). U50,488H relaxed the pulmonary artery rings of the hypoxic rats. This effect was partly abolished by l-NAME. In cells, U50,488H both increased NO production and reduced hypoxia-induced apoptosis. Moreover, pretreatment with nor-binaltorphimine (nor-BNI, a selective κ-opioid receptor antagonist), PI3K inhibitor, Akt inhibitor or l-NAME almost abolished anti-apoptotic effect exerted by U50,488H. U50,488H resulted in increases in Akt and eNOS phosphorylation. These results demonstrate that pretreatment with U50,488H attenuates hypoxia-induced pulmonary vascular endothelial dysfunction in an Akt-dependent and NO-mediated fashion.

Antiarrhythmic effect mediated by κ-opioid receptor is associated with Cx43 stabilization.

OBJECTIVE: Acute myocardial ischemia induces electrical and chemical uncoupling of gap junctions, which contributes to conduction abnormalities and re-entrant arrhythmias. We tested the hypothesis that structure and function of Connexin43 may vibrate during acute myocardial ischemia and reperfusion and κ-opioid receptor stimulation may stabilize the alteration of Connexin43. DESIGN: An animal intervention study was conducted with comparison to a control group. SETTING: University preclinical research laboratory. SUBJECTS: Age-, weight-, and sex-matched Sprague-Dawley rats. INTERVENTIONS: Adult rat hearts were subjected to ischemia or ischemia/reperfusion, which was induced by temporary occlusion of the left main coronary artery. U50488H was given 10 mins before tissue specimens were taken or before ischemia (1.5 mg/kg, intravenous) and nor-BNI was given 15 mins before tissue specimens were taken or before ischemia (2 mg/kg, intravenous). Tissue samples came from left ventricular myocardium of the rat hearts. MEASUREMENTS AND MAIN RESULTS: Electrocardiogram, immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction were used to measure changes of arrhythmias, protein, and gene expression of Connexin43, respectively. κ-opioid receptor activation with U50 decreased arrhythmia in a model of myocardial ischemia and reperfusion. In normal hearts, immunohistochemical data showed reduced amount and lateralization of Connexin43 induced by κ-opioid receptor activation, whereas immunoblotting data demonstrated no significant changes between control and U50 group. During ischemia, however, Connexin43 protein underwent dephosphorylation and degradation, and Connexin43 mRNA was upregulated. These alterations were significantly attenuated on κ-opioid receptor stimulation. During ischemia and reperfusion, Connexin43 protein underwent dephosphorylation and degradation and recovered slowly during reperfusion. Activation of κ-opioid receptor accelerated recovery of phosphorylated and total Connexin43. CONCLUSIONS: In normal rat hearts, Connexin43 translocates from intercellular junctions to intracellular locations on κ-opioid receptor activation. In rat hearts experiencing acute myocardial ischemia and reperfusion, protein and gene expression of Connexin43 undergo vibration. This phenomenon is stabilized when κ-opioid receptor is activated and by the fact that κ-opioid receptor produces antiarrhythmic effects.

Modulation of intracellular calcium transient in response to beta-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness.

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Myocardial apoptosis and infarction after ischemia/reperfusion are attenuated by kappa-opioid receptor agonist.

BACKGROUND AND AIMS: It remains unclear whether U50488H (a selective kappa-opioid receptor agonist) produces anti-apoptotic effect during ischemia and reperfusion (I/R). Therefore, the effect of U50488H on myocardial apoptosis was investigated in the present study. METHODS: Rats were subjected to 45min coronary artery occlusion and 180min of reperfusion. U50488H (1.5mg/kg IV) was given prior to occlusion. Nor-Binaltorphimine (nor-BNI) (2mg/kg IV), a selective kappa-opioid receptor antagonist, was given 10min prior to U50488H. Cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay and in situ identification of nuclear DNA fragmentation. RESULTS: The ultrastructure injury of myocardium, myocardial infarct size, and plasma CK and LDH were reduced significantly with administration of U50488H before I/R, whereas the effects of U50488H were abolished by nor-BNI. DNA fragments were visualized by agarose electrophoresis, and clear DNA ladder formation was observed in myocardial tissue from hearts subjected to I/R. Administration of U50488H before ischemia exerted a significant anti-apoptotic effect as evidenced by markedly weaker DNA ladder formation. TUNEL staining showed U50488H treatment before I/R significantly reduced the percentage of apoptotic cells, which was blocked by 5-HD, a mitochondrial k(ATP) channel blocker. In accordance, U50488H treatment significantly inhibited I/R-induced elevated activities of caspase-3 and caspase-9. U50488H also produced an increase in Bcl-2 and a decrease in Bax protein expression in the I/R heart, and the anti-apoptotic effects of U50488H were all blocked by nor-BNI. CONCLUSIONS: U50488H reduces myocardial necrosis and apoptosis after I/R and activation of kappa-opioid receptor may mediate a role in U50488H-induced myocardial protection.

Distribution of kappa-opioid receptor in the pulmonary artery and its changes during hypoxia.

The present study evaluated the distribution of kappa-opioid receptors (kappa-ORs) in pulmonary arteries (PAs) in rats and investigated whether kappa-ORs are altered in PAs during hypoxia. An animal model of hypobaric/hypoxic pulmonary hypertension and a pulmonary artery smooth muscle cell (PASMC) model of hypoxia were utilized. Distribution of kappa-ORs was determined by fluorescence immunohistochemistry and changes in kappa-ORs expression in PAs and PASMCs were determined by fluorescence immunohistochemistry or Western blot techniques. The kappa-ORs were primarily distributed in the smooth muscle layer of the PAs and in the nucleus of PASMCs. The expression of the kappa-ORs were increased in PAs of rats subjected to hypoxia for 1-4 week (P < 0.01). Accordingly, the expression of kappa-ORs in PASMCs were also increased when subjected to hypoxia for 12-36 hr (P < 0.05). The present study has provided evidence for the first time of the precise location of kappa-ORs in PAs and PASMCs of rats and that hypoxia upregulates expression of kappa-ORs.

Effects of U50,488H on hypoxia pulmonary hypertension and its underlying mechanism.

The aim of the present study was to determine whether U50,488H, a selective kappa-opioid receptor agonist, inhibits the remodeling of the pulmonary artery (PA). In addition, changes in the concentrations of nitric oxide (NO), endothelin (ET) and angiotensin II (AngII) in hypoxic pulmonary hypertensive (HPH) rats were investigated to explore the mechanisms underlying the effects of U50, 488H on HPH. We found that intraperitoneal administration of U50,488H (every other day) during hypoxia depressed mean pulmonary arterial pressure (mPAP) and attenuated right ventricular pressure (RVP) and right ventricular hypertrophy, at the same time it inhibited remodeling of the PA compared with hypoxia for 2 wk. Moreover, U50,488H also inhibited proliferation of the pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia for 48 h in a dose-dependent manner. Compared with the 2 wk hypoxia group, U50,488H increased the concentration of NO and decreased the production of ET and AngII (P<0.01). In addition, acute intravenous administration of U50,488H after hypoxia for 4 wk decreased mPAP. Our results suggest that effects of anti-remodeling of the PA and anti-proliferation of the PASMC, and regulation of the vasomotor factors in both blood and pulmonary tissues of HPH rats may be critical mechanisms underlying the preventive and therapeutic effects of U50,488H in HPH rats.

Stimulation of kappa-opioid receptor reduces isoprenaline-induced cardiac hypertrophy and fibrosis.

The aim of the present study was to determine whether U50,488H (a selective kappa-opioid receptor agonist) inhibits cardiac hypertrophy and fibrosis induced by beta-adrenoceptor stimulation in a rat model. Cardiac hypertrophy and fibrosis were developed by intraperitoneal administration of isoprenaline (ip. 3.0 mg/kg/day,14 days). In the isoprenaline-treated group, heart weight and heart-to-body ratio increased significantly. Hypertrophic alterations were observed in light micrographs of tissue and transmission electron micrographs of myocardial ultra structures. Increases in heart weight, heart-to-body ratio, diameter of cardiomyocytes, and morphological hypertrophic alterations induced by isoprenaline were significantly attenuated by U50,488H(i.p. 1.25 mg/kg/day). Growth of cardiomyocytes was induced by incubating with isoprenaline (10(-6) mol/l), which resulted in an increase in optical density (OD) values. The increased OD value was attenuated by U50,488H(10(-7) mol/l-10(-5) mol/l) in a dose dependent manner. Animals receiving administration of isoprenaline displayed significant fibrosis. The extent of isoprenaline induced left ventricular fibrosis was dramatically reduced in U50,488H treated animals. Increased cardiac fibroblast proliferation and collagen synthesis induced by isoprenaline, as evidenced by increased OD value, (3)H-thymidine, and (3)H-proline incorporation, were significantly reduced in the U50,488H treated group. The specific extracellular matrix proteins, including type I, type III collagen and fibronectin, which increased after administration of isoproterenol, were also attenuated by U50,488H. The abovementioned effects of U50,488H were completely abolished by nor-BNI (nor-binaltorphimine), a selective kappa-opioid receptor antagonist. The enhanced intracellular Ca(2+) transient and L-type Ca(2+) current elicited by isoprenaline in cardiomyocytes were significantly inhibited by U50,488H. This study provides the first morphological evidence of the inhibitory effect of U50,488H on isoprenaline-induced cardiac hypertrophy and fibrosis via kappa-opioid receptor stimulation.

Modulation of {beta}-adrenoceptor signaling in the hearts of 4-wk simulated weightlessness rats.

The modulation of beta-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on beta-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (-dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to beta-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and +/-dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and +/-dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsalpha (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes beta-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.